once again thanks for the response,
I'm sorry I think I didn't make myself clear before..
If I isolated the RNA of the sample --> then I synthesize the cDNA --> real-time PCR step,
so I have to do the same path with the standard (cell line), right?
I have to isolate the RNA of the cell line --> then I synthesize the cell line's cDNA --> measure its concentration by spectrophotometry --> the result (ng/ul) will be used to find out its copy numbers/ul (with Avogadro) --> making serial dilutions of the cell line --> the log copy numbers will become the x-axis of the standard curve.
(And then come the normalisation steps....)
Are those steps above right?
And about why I intend to know the RNA copy numbers of the sample, yes you're right, I want to know the number of specific RNAs in the sample,
because the principle investigator of this research wants to know the difference of RNA copy numbers of sample before
some treatme nts.
That's why I'm going to do the absolute quantification real-time PCR.
I feel challenged with this research but because I've never done it before I think I have to ask somewhere
, so that I'm sure that the methods I'm going to do is clear.
Moreover the absolute quantification depends on the 'goodness' of the standard curve, so this step is very crucial I think.
Thank you, again